We tested the efficacy of lapatinib, a dual tyrosine kinase inhibitor which interrupts the HER2 and epidermal growth factor receptor (EGFR) pathways, in a panel of triple-negative breast cancer (TNBC) cells, and examined the drug mechanism. Lapatinib showed an anti-proliferative effect in HCC 1937, MDA-MB-468, and MDA-MB-231 cell lines. Lapatinib induced significant apoptosis and inhibited CIP2A and p-Akt in a dose and time-dependent manner in the three TNBC cell lines. Overexpression of CIP2A reduced lapatinib-induced apoptosis in MDA-MB-468 cells. In addition, lapatinib increased PP2A activity (in relation to CIP2A inhibition). Moreover, lapatinib-induced apoptosis and p-Akt downregulation was attenuated by PP2A antagonist okadaic acid. Furthermore, lapatinib indirectly decreased CIP2A transcription by disturbing the binding of Elk1 to the CIP2A promoter. Importantly, lapatinib showed anti-tumor activity in mice bearing MDA-MB-468 xenograft tumors, and suppressed CIP2A as well as p-Akt in these xenografted tumors. In summary, inhibition of CIP2A determines the effects of lapatinib-induced apoptosis in TNBC cells. In addition to being a dual tyrosine kinase inhibitor of HER2 and EGFR, lapatinib also inhibits CIP2A/PP2A/p-Akt signaling in TNBC cells.

Chronic Obstructive Pulmonary Disease (COPD) is one of the fastest growing causes of death worldwide. However, few studies, if any, have been conducted that have investigated patient profiles in Asia. This paper analyzes patient willingness to pay (WTP) as a function of patient disease severity, health-related quality of life (HRQL), and smoking behavior in Taiwan.

Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC), a human malignancy notorious for its highly metastatic nature. Among EBV-encoded genes, latent membrane protein 1 (LMP1) is expressed in most NPC tissues and exerts oncogenicity by engaging multiple signaling pathways in a ligand-independent manner. LMP1 expression also results in actin cytoskeleton reorganization, which modulates cell morphology and cell motility- cellular process regulated by RhoGTPases, such as Cdc42. Despite the prominent association of Cdc42 activation with tumorigenesis, the molecular basis of Cdc42 activation by LMP1 in NPC cells remains to be elucidated. Here using GST-CBD (active Cdc42-binding domain) as bait in GST pull-down assays to precipitate active Cdc42 from cell lysates, we demonstrated that LMP1 acts through its transmembrane domains to preferentially induce Cdc42 activation in various types of epithelial cells, including NPC cells. Using RNA interference combined with re-introduction experiments, we identified FGD4 (FYVE, RhoGEF and PH domain containing 4) as the GEF (guanine nucleotide exchange factor) responsible for the activation of Cdc42 by LMP1. Serial deletion experiments and co-immunoprecipitation assays further revealed that ectopically expressed FGD4 modulated LMP1-mediated Cdc42 activation by interacting with LMP1. Moreover, LMP1, through its transmembrane domains, directly bound FGD4 and enhanced FGD4 activity toward Cdc42, leading to actin cytoskeleton rearrangement and increased motility of NPC cells. Depletion of FGD4 or Cdc42 significantly reduced (∼50%) the LMP1-stimulated cell motility, an effect that was partially reversed by expression of a constitutively active mutant of Cdc42. Finally, quantitative RT-PCR and immunohistochemistry analyses showed that FGD4 and LMP1 were expressed in NPC tissues, supporting the potential physiologically relevance of this mechanism in NPC. Collectively, our results not only uncover a novel mechanism underlying LMP1-mediated Cdc42 activation, namely LMP1 interaction with FGD4, but also functionally link FGD4 to NPC tumorigenesis.

The kallikrein/kinin system is beneficial in ischemia/reperfusion injury in heart, controversial in brain, but detrimental in lung, liver, and intestine. We examined the role of the kallikrein/kinin system in acute ischemia/reperfusion renal injury induced by 40 min occlusion of the renal artery followed by reperfusion. Rats were infused with tissue kallikrein protein 5 days before (pretreated group) or after (treated group) ischemia. Two days later, the pretreated group exhibited the worst renal dysfunction, followed by the treated group, then the control group. Kallikrein increased tubular necrosis and inflammatory cell infiltration with generation of more tumor necrosis factor-alpha and monocyte chemoattractant protein-1. Reactive oxygen species (ROS), malondialdehyde, and reduced/oxidized glutathione measurement revealed that the oxidative stress was augmented by kallikrein administration in both ischemic and reperfusion phases. The groups with more ROS generation also had more apoptotic renal cells. The deleterious effects of kallikrein on ischemia/reperfusion injury were reversed by cotreatment with bradykinin B2 receptor (B2R) antagonist, but not B1 receptor antagonist, and were not associated with hemodynamic changes. We conclude that early activation of B2R augmented ROS generation in ischemia/reperfusion renal injury, resulting in subsequent apoptosis, inflammation, and tissue damage. This finding suggests the potential application of B2R antagonists in acute ischemic renal disease associated with bradykinin activation.

Background: Urinary calprotectin is a novel biomarker that distinguishes between intrinsic or prerenal acute kidney injury (AKI) in different studies. However, these studies were based on different populations and different AKI criteria. We evaluated the diagnostic accuracy of urinary calprotectin and compared its diagnostic performance in different AKI criteria and study populations. Method: In accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we searched PubMed, Embase, and the Cochrane database up to September 2018. The diagnostic performance of urinary calprotectin (sensitivity, specificity, predictive ratio, and cutoff point) was extracted and evaluated. Result: This study included six studies with a total of 502 patients. The pooled sensitivity and specificity were 0.90 and 0.93, respectively. The pooled positive likelihood ratio (LR) was 15.15, and the negative LR was 0.11. The symmetric summary receiver operating characteristic (symmetric SROC) with pooled diagnostic accuracy was 0.9667. The relative diagnostic odds ratio (RDOC) of the adult to pediatric population and RDOCs of different acute kidney injury criteria showed no significant difference in their diagnostic accuracy. Conclusion: Urinary calprotectin is a good diagnostic tool for the discrimination of intrinsic and prerenal AKI under careful inspection after exclusion of urinary tract infection and urogenital malignancies. Its performance is not affected by different AKI criteria and adult or pediatric populations.

Rationally designed cationic phospha-polyaromatic fluorophores were prepared through intramolecular cyclization of the tertiary ortho-(acene)phenylene-phosphines mediated by CuII triflate. As a result of phosphorus quaternization, heterocyclic phosphonium salts 1 c-3 c, derived from naphthalene, phenanthrene, and anthracene cores, exhibited very intense blue to green fluorescence (Φem =0.38-0.99) and high photostability in aqueous medium. The structure-emission relationship was further investigated by tailoring the electron-donating functions to the anthracene moiety to give dyes 4 c-6 c with charge-transfer character. The latter significantly decreases the emission energy to reach near-IR region. Thus, the intramolecular phosphacyclization renders an ultra-wide tuning of fluorescence from 420 nm (1 c) to 780 nm (6 c) in solution, extended to 825 nm for 6 c in the solid state with quantum efficiency of approximately 0.07. The physical behavior of these new dyes was studied spectroscopically, crystallographically, and electrochemically, whereas computational analysis was used to correlate the experimental data with molecular electronic structures. The excellent stability, water solubility, and attractive photophysical characteristics make these phosphonium heterocycles powerful tools in cell imaging.

Vascular endothelial growth factor (VEGF) is correlated with angiogenesis and early relapse of colorectal cancer (CRC). This study investigated the role of miR-148a in the regulation of VEGF/angiogenesis and early relapse of CRC. We established a stable clone with miR-148a expression in HCT116 and HT29 cell lines and created a hypoxic condition by using CoCl2 to determine the underlying mechanism of miR-148a. The effects of miR-148a on the phosphoryl-ERK (pERK)/hypoxia-inducible factor-1α (HIF-1α)/VEGF pathway were evaluated through Western blotting and the inhibitory effect of miR-148a on angiogenesis was demonstrated through a tube formation assay. Sixty-three CRC tissues (28 early relapse and 35 non-early relapse) were analysed to assess the relationship between miR-148a and HIF-1α/VEGF. The protein expression of pERK/HIF-1α/VEGF in HCT116 and HT29 cells was significantly decreased by miR-148a (all P < 0.05). The protein expression of VEGF/HIF-1α was strongly inversely associated with the expression of miR-148a in the 63 CRC tissue samples (all P < 0.05). Tube formation assay demonstrated that miR-148a significantly obliterated angiogenesis. miR-148a suppresses VEGF through down-regulation of the pERK/HIF-1α/VEGF pathway and might lead to the inhibition of angiogenesis; miR-148a down-regulation increased the early relapse rate of CRC. This demonstrates that miR-148a is a potential diagnostic and therapeutic target.

Triple negative breast cancer (TNBC) lacks specific drug targets and remains challenging. Palbociclib, a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor is approved for metastatic estrogen receptor (ER)-positive and human epithermal growth factor 2 (HER2)-negative breast cancer. The nature of cell cycle inhibition by palbociclib suggests its potential in TNBC cells. Retinoblastoma (RB, a known substrate of CDK4/6) pathway deregulation is a frequent occurrence in TNBC and studies have revealed that pharmacological CDK4/6 inhibition induces a cooperative cytostatic effect with doxorubicin in RB-proficient TNBC models. In addition, recent studies reported that anti-androgen therapy shows preclinical efficacy in androgen-receptor (AR)-positive TNBC cells. Here we examined the effect of palbociclib in combination with an anti-androgen enzalutamide in TNBC cells.

Patients with pneumonia and parapneumonic effusion (PPE) have elevated mortality and a poor prognosis. The aim of this study was to discover novel biomarkers to help distinguish between uncomplicated PPE (UPPE) and complicated PPE (CPPE). Using an iTRAQ-based quantitative proteomics, we identified 766 proteins in pleural effusions from PPE patients. In total, 45 of these proteins were quantified as upregulated proteins in CPPE. Four novel upregulated candidates (BPI, NGAL, AZU1, and calprotectin) were selected and further verified using enzyme-linked immunosorbent assays (ELISAs) on 220 patients with pleural effusions due to different causes. The pleural fluid levels of BPI, NGAL, AZU1, and calprotectin were significantly elevated in patients with CPPE. Among these four biomarkers, BPI had the best diagnostic value for CPPE, with an AUC value of 0.966, a sensitivity of 97%, and a specificity of 91.4%. A logistic regression analysis demonstrated a strong association between BPI levels > 10 ng/ml and CPPE (odds ratio = 341.3). Furthermore, the combination of pleural fluid BPI levels with LDH levels improved the sensitivity and specificity to 100% and 91.4%, respectively. Thus, our findings provided a comprehensive effusion proteome data set for PPE biomarker discovery and revealed novel biomarkers for the diagnosis of CPPE.

Patterns of genetic alterations characterize different molecular subtypes of human gastric cancer. We aimed to establish mouse models of these subtypes.

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway that modulates cellular redox homeostasis via the regeneration of NADPH. G6PD-deficient cells have a reduced ability to induce the innate immune response, thus increasing host susceptibility to pathogen infections. An important part of the immune response is the activation of the inflammasome. G6PD-deficient peripheral blood mononuclear cells (PBMCs) from patients and human monocytic (THP-1) cells were used as models to investigate whether G6PD modulates inflammasome activation. A decreased expression of IL-1β was observed in both G6PD-deficient PBMCs and PMA-primed G6PD-knockdown (G6PD-kd) THP-1 cells upon lipopolysaccharide (LPS)/adenosine triphosphate (ATP) or LPS/nigericin stimulation. The pro-IL-1β expression of THP-1 cells was decreased by G6PD knockdown at the transcriptional and translational levels in an investigation of the expression of the inflammasome subunits. The phosphorylation of p38 MAPK and downstream c-Fos expression were decreased upon G6PD knockdown, accompanied by decreased AP-1 translocation into the nucleus. Impaired inflammasome activation in G6PD-kd THP-1 cells was mediated by a decrease in the production of reactive oxygen species (ROS) by NOX signaling, while treatment with hydrogen peroxide (H2O2) enhanced inflammasome activation in G6PD-kd THP-1 cells. G6PD knockdown decreased Staphylococcus aureus and Escherichia coli clearance in G6PD-kd THP-1 cells and G6PD-deficient PBMCs following inflammasome activation. These findings support the notion that enhanced pathogen susceptibility in G6PD deficiency is, in part, due to an altered redox signaling, which adversely affects inflammasome activation and the bactericidal response.

Uncoupling protein 2 (UCP2) may be critical for intestinal barrier function which may play a key role in the development of sepsis, and insulin has been reported to have anti-inflammatory effects. Male Sprague-Dawley rats were randomly allocated into five groups: control group, cecal ligation and puncture (CLP) group, sham surgery group, CLP plus glucose-insulin-potassium (GIK) group, and CLP plus glucose and potassium (GK) group. Ileum tissues were collected at 24 h after surgery. Histological and cytokine analyses, intestinal permeability tests, and western blots of intestinal epithelial tight junction component proteins and UCP2 were performed. Compared with CLP group, the CLP + GIK group had milder histological damage, lower levels of cytokines in the serum and ileum tissue samples, and lower UCP2 expression, whereas the CLP + GK group had no such effects. Moreover, the CLP + GIK group exhibited decreased epithelial permeability of the ileum and increased expression of zonula occludens-1, occludin, and claudin-1 in the ileum. The findings demonstrated that the UCP2 and NLR family-pyrin domain-containing 3/caspase 1/interleukin 1β signaling pathway may be involved in intestinal barrier injury and that GIK treatment decreased intestinal barrier permeability. Thus, GIK may be a useful treatment for intestinal barrier injury during sepsis.

The role of fibroblasts in tissue fibrosis has been extensively studied. Activated fibroblasts, namely myofibroblasts, produce pathological extracellular matrix. CD248, a type I transmembrane glycoprotein, is expressed in fibroblasts after birth. In human chronic kidney disease, upregulated CD248 in myofibroblasts is linked to poor renal survival. In this study, we demonstrated a novel interaction between CD248 and macrophages to be a key step in mediating tissue fibrosis. CD248 was upregulated in myofibroblasts in murine models of renal and peritoneal fibrosis. Cd248 knockout (Cd248-/-) could attenuate both renal and peritoneal fibrosis. By parabiosis of GFP reporter mice and Cd248-/- mice, we showed that attenuation of renal fibrosis was associated with a decrease of macrophage infiltration in Cd248-/- mice. Moreover, decrease of chemokine (C-C motif) ligand 17 and Ccl22 was found in macrophages isolated from the fibrotic kidneys of Cd248-/- mice. Because galectin-3-deficient macrophages showed decreased Ccl17 and Ccl22 in fibrotic kidneys, we further demonstrated that CD248 interacted specifically with galectin-3 of macrophages who then expressed CCL17 to activate collagen production in myofibroblasts. Mice with DNA vaccination targeting CD248 showed decreased fibrosis. We thus propose that CD248 targeting should be studied in the clinical tissue fibrosis setting.

Colorectal cancer (CRC) of the clinical tumor stage T4b (cT4b) refers to advanced tumors with direct invasion of adjacent structures and the tumors are considered unresectable. Despite advancements in aggressive surgery and combination chemotherapy, the prognosis of cT4b CRC remains poor. Optimizing the therapeutic sequence administered to patients with cT4b CRC to improve clinical outcomes is crucial. In the present study, patients with unresectable cT4b and nodal stage N1-2 CRC were investigated at a single institution. A total of 20 consecutive patients were treated with pre-operative concurrent chemoradiation by using 5-fluorouracil/leucovorin/oxaliplatin (FOLFOX) since February 2015 and were regularly followed up until March 2020. Due to their poor response to concurrent chemoradiation (CCRT) with FOLFOX, the chemotherapy regimen was changed to irinotecan plus 5-fluorouracil/leucovorin (FOLFIRI) as the second-line neoadjuvant treatment. Genetic alterations, such as microsatellite instability (MSI), were documented, and the expression levels of excision repair cross-complementing group 1 (ERCC1) and ERCC2 were examined. Of the 20 patients, the tumors of 14 patients (70%) became resectable after FOLFIRI administration. The median duration between the last date of radiotherapy and surgery was 32.7 weeks (range, 10.1-59.3 weeks). Of note, 4 of the 14 patients with resectable tumors (28.6%) achieved a pathologic complete response. The median overall survival and progression-free survival were 27.5 months (range, 12-39 months) and 27.5 months (range, 8-39 months), respectively. The cancerous specimens of all of the patients (100%) exhibited ERCC2 overexpression and 18 specimens (90%) had ERCC1 overexpression. Only one tumor (5%) exhibited high MSI. The present study indicated that ERCC overexpression associated with the poor response of FOLFOX-based CCRT and FOLFIRI after FOLFOX-based CCRT failure may have a potential role in conversion to resectable tumors by neoadjuvant treatment in cT4b CRC. However, a further prospective study with more patients is required to improve the precision of the conclusions.

Aquaporin-2 (AQP2) is a molecular water channel protein responsible for water reabsorption by the kidney collecting ducts. Many water balance disorders are associated with defects in AQP2 gene expression regulated by the peptide hormone vasopressin. Here, we studied roles of Elf3 (E26 transformation-specific (Ets)-related transcription factor 3) in AQP2 gene expression in the collecting duct cells (mpkCCD). Vasopressin increased AQP2 mRNA and protein levels without affecting AQP2 mRNA degradation, indicative of transcriptional regulation. Elf3 knockdown and overexpression, respectively, reduced and increased AQP2 gene expression under basal and vasopressin-stimulated conditions. However, the vasopressin-to-basal ratios of AQP2 gene expression levels remained constant, indicating that Elf3 does not directly mediate vasopressin response but modulates the level of AQP2 gene expression inducible by vasopressin. The Elf3-modulated AQP2 gene expression was associated with AQP2 promoter activity, in line with Elf3's ability to bind an Ets element in the AQP2 promoter. Mutation in the Ets element reduced both basal and vasopressin-stimulated AQP2 promoter activity, again without affecting vasopressin-to-basal ratios of the AQP2 promoter activity. Lithium chloride reduced both Elf3 and AQP2 mRNA in the mpkCCD cells as well as in mouse kidney inner medulla. We conclude that Elf3 modulates AQP2 promoter activity thereby gauging vasopressin-inducible AQP2 gene expression levels. Our data provide a potential explanation to lithium-induced nephrogenic diabetes insipidus where lithium reduces Elf3 and hence AQP2 abundance.

Antibacterial resistance (ABR) poses an enormous threat to human health. ABR mainly develops due to bacteria being constantly exposed to diluted levels of disinfectants. Here, we propose a method for suppressing ABR through the chemical binding of disinfectants to polymethyl methacrylate (PMMA) device surfaces in solutions of 5%, 10%, and 20% disinfectant concentrations. PMMA discs were fabricated from a commercial orthodontic acrylic resin system (Ortho-Jet) and quaternary ammonium salts (QAS), 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride (42% in methanol), were used as the disinfectant. The PMMA surfaces were activated in 3 M sulfuric acid at 80 °C for 5 h for the esterification of hydrolyzed QAS to PMMA. Fourier transform infrared difference spectra confirmed that the carboxy-terminated PMMA was chemically bound to the QAS. In vitro cell viability tests using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays revealed that 5%QAS-c-PMMA was more biocompatible than 10%QAS-c-PMMA and 20%QAS-c-PMMA. The results of antibacterial tests and clinical trials demonstrated the excellent antibacterial power of 5%QAS-c-PMMA. This method is the first solution-based approach to successfully avoid disinfectant leakage and subsequent ABR, as revealed by mass spectrometry studies of the solution obtained by agitating the disinfectant-bound PMMA for 28 days.

Epidermal growth factor receptor (EGFR) is frequently overexpressed and mutated in non-small cell lung cancer (NSCLC), which is the major type of lung cancer. The EGFR tyrosine kinase inhibitors (TKIs) are the approved treatment for patients harboring activating mutations in the EGFR kinase. However, most of the patients treated with EGFR-TKIs developed resistance. Therefore, the development of compounds exhibiting unique antitumor activities might help to improve the management of NSCLC patients. The total flavonoids from Daphne genkwa Sieb. et Zucc. have been shown to contain antitumor activity. Here, we have isolated a novel flavonoid hydroxygenkwanin (HGK) that displays selective cytotoxic effects on all of the NSCLC cells tested. In this study, we employed NSCLC cells harboring EGFR mutations and xenograft mouse model to examine the antitumor activity of HGK on TKI-resistant NSCLC cells. The results showed that HGK suppressed cancer cell viability both in vitro and in vivo. Whole-transcriptome analysis suggests that EGFR is a potential upstream regulator that is involved in the gene expression changes affected by HGK. In support of this analysis, we presented evidence that HGK reduced the level of EGFR and inhibited several EGFR-downstream signalings. These results suggest that the antitumor activity of HGK against TKI-resistant NSCLC cells acts by enhancing the degradation of EGFR.

The incidence of oral squamous cell carcinoma (OSCC), which is one of the most common cancers worldwide, has been increasing. Serum anti-p53 autoantibody is one of the most sensitive biomarkers for OSCC. Currently, the most commonly used method on clinical screening platforms is the enzyme-linked immunosorbent assay, owing to its high specificity and repeatability. However, conducting immunoassays on 96-well plates is typically time consuming, thereby limiting its clinical applications for fast diagnosis and immediate prognosis of rapidly progressive diseases. The present study performed immunoassays in glass capillaries of 1-mm internal diameter, which increases the surface to volume ratio of the reaction, to shorten the time needed for immunoassay. The immunoassay was automated while using linear motorized stages and a syringe pump. The results indicated that, when compared with the 96-well plate immunoassay, the glass capillary immunoassay decreased the reaction time from typical 120 min to 45 min, reduced the amount of reagent from typical 50 µL to 15 µL, and required only simple equipment setup. Moreover, the limit of detection for glass capillary anti-p53 autoantibody immunoassay was 0.46 ng mL-1, which is close to the 0.19 ng mL-1 value of the conventional 96-well plate assay, and the glass capillary method had a broader detection range. The apparatus was used to detect the serum anti-p53 autoantibody concentration in clinical patients and compare its results with the conventional 96-well plate method results, which suggested that both of the methods detect the same trend in the relative concentration of serum anti-p53 autoantibody in healthy individuals or patients with OSCC.

The development of Bretschneider's histidine-tryptophan-ketoglutarate (HTK) cardioplegia solution represented a major advancement in cardiac surgery, offering significant myocardial protection. However, metabolic changes induced by this additive in the whole body have not been systematically investigated. Using an untargeted mass spectrometry-based method to deeply explore the urine metabolome, we sought to provide a holistic and systematic view of metabolic perturbations occurred in patients receiving HTK. Prospective urine samples were collected from 100 patients who had undergone cardiac surgery, and metabolomic changes were profiled using a high-performance chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method. A total of 14,642 peak pairs or metabolites were quantified using differential 13C-/12C-dansyl labeling LC-MS, which targets the amine/phenol submetabolome from urine specimens. We identified 223 metabolites that showed significant concentration change (fold change > 5) and assembled several potential metabolic pathway maps derived from these dysregulated metabolites. Our data indicated upregulated histidine metabolism with subsequently increased glutamine/glutamate metabolism, altered purine and pyrimidine metabolism, and enhanced vitamin B6 metabolism in patients receiving HTK. Our findings provide solid evidence that HTK solution causes significant perturbations in several metabolic pathways and establish a basis for further study of key mechanisms underlying its organ-protective or potential harmful effects.

Twin pregnancies continue to increase worldwide; however, the current clinical prenatal evaluation for the intrauterine growth of twins still relies on the growth standards of singletons. We attempted to establish a set of fetal biometric references for Chinese twin pregnancies, stratified by chorionicity and conception mode as spontaneously conceived monochorionic diamniotic (SC-MCDA), spontaneously conceived dichorionic diamniotic (SC-DCDA), and assisted reproductive technology dichorionic diamniotic (ART-DCDA) twins.